42-oximes and hydroxylamine rapamycin, method of production, intermediate product, pharmaceutical composition and method of treatment

 

(57) Abstract:

Describes new compounds of formula I where X-Y is C=NR1or NR2in which R1denotes hydrogen, alkyl with 1 to 6 carbon atoms, alkenyl with 2 to 7 carbon atoms, quinil with 2 to 7 carbon atoms, a group of the formula -(CH2)mAr, in which Ar is phenyl or pyridinyl and m = 1 to 6; R2represents hydrogen or a radical of the formula -(CH2)mAr, in which Ar represents pyridinyl, or their pharmaceutically acceptable salts. Also describes the method of production thereof, the intermediate product, pharmaceutical composition and method of treatment of the disease. The new compounds are useful as immunosuppressive, antiinflammatory, antifungal, antiproliferative and anticancer agents. 5 C. and 13 C.p. f-crystals.

This invention relates to a 42-oximes and hydroxylamines rapamycin and to method of using them for inducing immunosuppression and in the treatment of transplant rejection, graft versus host disease, autoimmune diseases, inflammatory diseases, T-cell leukemia/lymphoma in adults, solid tumors, fungal infections or fungal infections, and hyperproliferative reptomyces hygroscopicus, which was discovered antifungal activity, particularly against Candida albicans, both in vitro and in vivo [C. Vezina et al., J. Antibiot. 28, 721 (1975); S. N. Sehgal et al., J. Antibiot. 28, 727 (1975); H. A. Baker et al., J. Antibiot. 31, 539 (1978); U.S. Patent 3929992 and U.S. Patent 3993749].

It has been shown that rapamycin, used one (one-component) (U.S. Patent 4885171) or in combination (combination) with picibanil (U.S. Patent 4401653), and antitumor activity. P. Martel and others (R. Martel et al.) [Can. J. Physiol. Pharmacol. 55, 48 (1977)] have found that rapamycin is effective when used in an experimental model of allergic encephalomyelitis model of multiple sclerosis, the model of adjuvant arthritis model of rheumatoid arthritis and inhibits or suppresses the formation of immunoglobulin E-like antibodies.

Immune (immune) effect of rapamycin was described in the FASEB 3, 3411 (1989). It was also shown that cyclosporine a and FK-506, other macrocyclic molecules effective as immunosuppressive funds and, thus, are useful in preventing transplant rejection [FASEB 3, 3411 (1989); FASEB 3, 5256 (1989); R. Y. Calne et al., Lancet 1183 (1978); U.S. Patent 5100899].

It was also shown that rapamycin is effective in preventing or is Chernogo diabetes [Fifth Int. Conf. Inflamm. Res. Assoc. 121 (Abstract), (1930)], the cell proliferation of smooth muscles and intimal thickening after vascular injury [Morris, R. J. Heart Lung Transplant 11 (pt. 2): 197 (1992)], T-cell leukemia/lymphoma [European patent application 525960 A1] and inflammation of the eyes (European patent application 532862 A1).

It has been shown that mono - diallylamine derivatives of rapamycin (esterified at positions 28 and 43) effective as antifungal agents (U.S. Patent 4316885) and can be used to obtain water-soluble aminoacylating prodrugs of rapamycin (U.S. Patent 4650803). It was recently modified agreement on the numbering of rapamycin, therefore, in accordance with the nomenclature of Chemical abstracts, education above esters should be considered in positions 31 and 42. In U.S. Patent 5023263 describes the collection and use of 42-oxalipatin, and in U.S. Patent 5023264 described the preparation and use 27-oximo rapamycin.

Description

The invention relates to a derivative of rapamycin that can be used as immunosuppressive, antiinflammatory, antifungal, antiproliferative, and protivoopuhulevyh means, having a structure

< / BR>
where X-Y is C=NOR1or CHNHOR2what Ohm carbon, quinil with 2-7 carbon atoms, aminoalkyl with 1-6 carbon atoms, acylaminoalkyl with 1-6 carbon atoms in each alkyl group, dialkylaminoalkyl with 1-6 carbon atoms in each alkyl group, cycloalkyl with 3-8 carbon atoms, alkyloxy with 1-6 carbon atoms, alkoxyalkyl with 1-6 carbon atoms in each alkyl group, cycloalkylation from 4-14 carbon atoms, cianelli with 2-7 carbon atoms, foralkyl with 1-6 carbon atoms, triptorelin with 2-7 carbon atoms, trifluoromethyl, -, -(CH2)mAr or-COR3;

R3denotes alkyl with 1-6 carbon atoms, -NH2-THE OTHER4, -NR4R5, -OR4or ,

each of R4and R5independently represents alkyl with 1-6 carbon atoms, Ar, or, in the case of both, they can be taken together with the formation of a 4-7-membered ring;

Ar denotes aryl or heteroaryl radical, which may be optionally mono-, di - or tizamidine group selected from alkyl with 1-6 carbon atoms, alkenyl with 2-7 carbon atoms, quinil with 2-7 carbon atoms, arylalkyl with 7-10 carbon atoms, alkoxy with 1-6 carbon atoms, cyano, halogen, hydroxy, nitro, carbalkoxy with 2-7 carbon atoms, trifloromethyl, triptime the kind hydroxyalkyl with 1-6 carbon atoms, alkoxyalkyl with 2-12 carbon atoms, alkylthio with 1-6 carbon atoms, -SO3H and-CO2H; m = 0-6;

or their pharmaceutically acceptable salts.

Pharmaceutically acceptable salts are salts formed from such inorganic cations such as sodium, potassium, etc., and organic bases, such as mono-, di - and trialkylamines with 1-6 carbon atoms in each alkyl group and mono-, di - and trihydroxystilbene with 1-6 carbon atoms in each alkyl group, etc., and organic and inorganic acids such as: acetic, lactic, citric, tartaric, succinic, maleic, malonic, gluconic, hydrochloric, Hydrobromic, phosphoric, nitric, sulphuric acid, methanesulfonate and others, also known acceptable acids.

Terms: alkyl with 1-6 carbon atoms, alkenyl with 2-7 carbon atoms and quinil with 2-7 carbon atoms include non-branched chain or branched carbon chain. Examples of the alkyl as a group or part of a group, for example, arylalkyl, alkoxy or carbalkoxy are unbranched or branched chain with 1 to 6 carbon atoms, preferably 1-4 carbon atoms, for example methyl, ethyl, propyl, isopropy therefore, this invention encompasses not only a mixture of geometric isomers, but individual E and Z isomers which can be separated by methods known to experts in this field. Similarly, hydroxylamine of the present invention are composed of a mixture of epimeres at C-42, and therefore, this invention encompasses not only a mixture of isomers and individual isomers which can be separated by methods known to experts in this field. The term "aryl" as a group or part of a group, for example, arylalkyl includes any mono - or beerbottles aromatic group with 6-10 carbon atoms. The term "heteroaryl" includes any mono - or bicyclic heterocyclic aromatic radical comprising from 5 to 10 atoms in the heterocycle, of which up to three atoms in the heterocycle, the same or different, are heteroatoms selected from atoms of oxygen, nitrogen and sulfur.

Aryl and heteroaryl radicals Ar are preferably phenyl, pyridyl, furyl, pyrrolyl, thiophenyl, imidazolyl, oxazolyl or thiazolyl, which may not necessarily be mono-, di - or tizamidine group selected from alkyl with 1-6 carbon atoms, alkenyl with 2-7 carbon atoms, quinil with 2-7 carbon atoms, arylalkyl with 7-10 carbon atoms, alkoxy with 1-6 atoms, the Mino, dialkylamino with 1-6 carbon atoms in each alkyl group, dialkylaminoalkyl with 3-12 carbon atoms, hydroxyalkyl with 1-6 carbon atoms, alkoxyalkyl with 2-12 carbon atoms, alkylthio with 1-6 carbon atoms, -SO3H and CO2N.

If R3refers to-NR4R5then the group R4and R5may be the same or different (as defined above) or may be combined with the formation of saturated heterocycle consisting of 4 to 7 atoms in the ring which 1 atom is nitrogen and 0-2 other atom in the heterocycle may be nitrogen atoms, oxygen or sulfur. In the case when R4and R5taken together, it is preferable that R4R5was a carbon chain forming azetidinone, pyrolidine, piperidine or homopiperazine ring.

Preferred compounds of the present invention are compounds in which X-Y denotes C=NOR1; compounds in which X-Y denotes C= NOR1and R1denotes hydrogen, alkyl with 1-6 carbon atoms, alkenyl with 2-7 carbon atoms, quinil with 2-7 carbon atoms, alkyloxy with 1-6 carbon atoms, -(CH2)mAr or-COR3; compounds in which X-Y denotes CHNHOR

The present invention also describes methods of preparing compounds of rapamycin according to the present invention. In particular, this invention describes methods of obtaining a 42-oximo and hydroxylamino derivatives of rapamycin, which include one of the following methods:

a) interaction 42-oxalipatin (optionally protected hydroxyl functional group in position 31 silyl group of the formula R6defined below) with a hydroxylamine of the formula:

H2NOR1,

where R1has the above values, except those containing carbonyl parts with the formation of the corresponding oxime of the formula I, where X-Y denotes C=NOR1and destruction of specified protective group, R6,

or

b) the restoration of the oxime of the formula I (optionally protected hydroxyl functional group in position 31 silyl group of the formula R6defined below), where X-Y denotes C=NOR1where R1has the above values, except for those containing carbonyl radicals, with the formation of a hydroxylamine of the formula I, where X-Y denotes CHNHOR2,

or

C) the interaction of the compounds of formula I (optionally protected hydroxyl functional group, R1denotes hydrogen, cyanate, for example, alkali metal cyanate such as sodium cyanate, with the formation of the compounds of formula I, where X-Y denotes C=NOR1where R1represents-CONH2,

or

g) the interaction of the compounds of formula I, where X-Y denotes C=NOR1or CHNHOR2where R1and R2are hydrogen (where X-Y denotes C=NOR1not necessarily protected hydroxyl functional group in position 31 silyl group of the formula R6defined below) with an isocyanate or halogenosilanes formula

R4NCO, R4NCO or R4R5NCO,

where hal represents halogen, such as chlorine with formation of the corresponding compounds of formula I in which R1or R2denote COR3and R3means other4or NR4R5,

or

d) the interaction of the compounds of formula I, where X-Y denotes C=NOH or CHNHOH (where X-Y denotes C=NOH, optionally protected on the 31st hydroxyl functional group, silyl group of the formula R6defined below), alkyl - or arillotta with getting carbonate of the formula I, where R1or R2denotes COR3where R3denotes alkyl or aryl is ü obtained with a moderate output through selective (selective) oxidation of rapamycin 42-position by means of oxidation, mediated by ruthenium as claimed in the U.S. Patent 5023263 listed here for information. Alternatively, about 50% exit 42-oxalipatin can be obtained using a mixture of perruthenate of tetrapropylammonium/N-oxide N - methylmorpholine, as indicated Holt in PCT Publication U.S. 93/0668. Then the 42-oxalipatin can be processed appropriately substituted hydroxylamine to obtain a mixture of 42(E) and (Z) Asimov (X-Y is C=NOR1), which can be separated by standard procedures. Next, a 42-oximes can enter into reaction with a suitable reducing agent, for example, with a mixture of cyanoborohydride sodium /THF/ dioxane/pH 3.5 with education hydroxyamino of the present invention (X-Y is CHNHOR2).

Compounds of the present invention, in which X-Y denotes C=NOR1and R2means containing the carbonyl portion of the molecule, can be obtained from the 42-oxime of rapamycin (X-Y is C=NOR1and R1is hydrogen). For example, the 42-oxime rapamycin can react with sodium cyanate to form compounds in which R1denotes COR3and R3denotes NH2. Similarly, the 42-oxime rapamycin can be processed appropriately replacing the R1means-COR3and R3refers to-NR4R5. Similarly, you can get a connection that1is-COR3and R3is-other4. In addition, the processing of 42-oxime rapamycin corresponding alkyl - or arillotta in pyridine and a solvent such as methylene chloride, allows to obtain the oxime carbonates, in which R1is-COR3and R3is alkyl or Ar.

Similar functionalization can be achieved when X-Y is CHNHOR2using the parent compound, in which R1is hydrogen (obtained by restoring the 42-oxime of rapamycin by cyanoborohydride).

Alternatively, oxidative method for producing a 42-oxalipatin using ruthenium, this invention describes a synthetic route for obtaining Akimov and hydroxylamine according to the present invention by oxidation periodinane dess-Martin 31-O-protected rapamycin. Protection of the 31-position of rapamycin has been described in U.S. Patent 5120842 included here for information. For example, treatment of rapamycin with suitable protective reagent such as a mixture of triethylsilyl triflate /2,6-lutidine/ MENA. Oxidation periodinane dess-Martin provides approximately 65% yield 31-O-triethylsilyl-42-oxalipatin, which can be subjected to further processing of the appropriate hydroxylamine (and then the subsequent derivatives), as described above. For oximo of the present invention triethylsilyl protective group can be removed under conditions of low acidity (acetic acid /THF/ water), as described in U.S. Patent 5120842. When using this method hydroxylamine of the present invention can also be obtained from the appropriate Asimov restoring cyanoborohydride in an acidic environment with simultaneous removal of the silyl protective group.

Based on the above methodology dess-Martin using periodinane following compounds are intermediates useful for the preparation of Asimov of the present invention.

< / BR>
where R6means-SiR7R8R9and each of R7, R8and R9independently represents alkyl with 1-8 carbon atoms, alkenyl with 1-8 carbon atoms, phenylalkyl with 7-10 carbon atoms, triphenylmethyl or phenyl.

The compound of example 1 is the preferred promezhutochn obtain an intermediate product of the formula II, described above, which consists in the oxidation of 31-O-protected rapamycin (where the protecting group is R6defined above) under conditions suitable for carrying out oxidation dess-Martin periodinane and, if desired, removing the above-mentioned protective group.

The reagents used to produce compounds of the present invention, can be either purchased or obtained by using the standard methods described in the literature.

Immunosuppressive activity of typical compounds of the present invention were evaluated using standard methods of pharmacological in vitro tests to measure the degree of suppression of lymphocyte proliferation (factor lymphocyte activation/LAF/) and two standard procedures pharmacological testing in vivo. Using test procedures using a piece of skin graft measure the immunosuppressive activity of the test compound and the ability of test compounds to inhibit (inhibit) or to treat graft rejection. Using the standard pharmacological test using adjuvant arthritis measure the ability of test compounds to inhibit getnow methodology pharmacological trials of rheumatoid arthritis. Procedure the data in standard ways pharmacological tests described below.

Procedure proliferation of thymocytes caused by somitogenesis (LAF) was used to assess the immunosuppressive actions of typical compounds in vitro. Briefly, cells in the thymus of normal mice of BALB/c mice were cultured for 72 h with PHA and interleukin-1 and subjected to pulse tritium-labeled thymidine during the last 6 hours Cells were cultured with various concentrations of rapamycin, cyclosporine or test compounds or without them. Collect cells and determine the incorporated radioactivity. Suppression of lymphoproliferative estimated as the percentage change in the number of pulses per minute, compared to control groups who did not receive the drug. In order to compare in parallel with the evaluation of the effects of each compound was evaluated the effect of rapamycin. Figure IC50was obtained for each test compound, and rapamycin. When evaluating rapamycin as agents for comparison with typical compounds of the present invention, its indicator IC50was in the range of from 0.5 to 3.3 nanomoles. The obtained results of rezultaty, obtained for typical compounds of the present invention, expressed as the ratio, in comparison with rapamycin. A positive ratio indicates immunosuppressive activity. If the ratio is greater than one, this means that when using the test connection, the degree of inhibition of proliferation of thymocytes was higher than when using rapamycin. Calculation of the relationship is shown below.

< / BR>
Typical compounds of the present invention was also evaluated by the method of in vivo tests designed to determine the survival time (life) pieces of skin grafts transplanted from donor males line BALA/c males treated with C3H(H-2K). This method was borrowed from Billingham R. T. and Medawar, P. C., J. Exp. Biol. 28: 385-402, (1951). Briefly, skin transplantat in the form of pieces received from the donor was transplanted on the back of the recipient as an allograft, and istranslated was used as control in the same area. Recipients were injected intraperitoneally or orally with various concentrations of test compounds. Rapamycin was used as control. Recipients who have not received the test compounds were used as control for ottoreinsprecht did not dry up and not turned into a blackened crust. This moment was the day of rejection. Average time viability of transplant (number of days standard deviation) in the group treated with the test compound, compared with the figures obtained in the control group. The results are expressed as the average time the viability of the transplant in days. Kulichkova grafts in not receiving the test compound (control) group were usually rejected within 6-7 days. Compounds were tested at a dose of 4 mg/kg, administered intraperitoneally. Intraperitoneal injection of rapamycin at a dose of 4 mg/kg time the viability of the graft was 11,6710,63 day.

In standard pharmacological test using adjuvant arthritis assess the ability of test compounds to prevent immunopositive inflammation and suppress or cure rheumatoid arthritis. The following summarizes the methodology used in testing. Connection pre-injected rats (inbred rats male Wistar rats Lewis) for testing (for 1 h before injection of the antigen), and then in the right hind paw of these rats were injected full beta-blockers (F) to induce arthritis. Then certain doses were administered to rats pen is adnie feet measured on the 16th, 23rd and 30th day. Determined the difference in the volume of the paws (ml) from 16 to 0 days and expected percentage change compared with control. Inflammation of the left rear foot (not subjected to injection) caused by inflammation mediated by T-cells, and is recorded as the percentage change compared with control. On the other hand, inflammation of the right hind legs due to nonspecific inflammation. Compounds were tested at a dose of 2 mg/kg the Results are presented as percentage changes in the foot, not subjected to injection, on the 16th day compared with the control, the more negative percentage change, the better the connection. Introduction rapamycin provided 70% change compared to control. This means that in rats that received rapamycin was observed in 70% less immunopositive inflammation, than rats in the control group.

The results of these standard pharmacological tests, are presented after the description of the method for obtaining specific compounds which have been tested.

The results of these standard pharmacological tests testify to the immunosuppressive action of the compounds according to the present izobretatelstvo, thus, the immunosuppressive activity of the compounds of the present invention. The effectiveness of the compounds of the present invention as immunosuppressive substances was further proved by the results obtained in the standard pharmacological tests using skin grafts and adjuvant arthritis. In addition, the results obtained in the test using a skin graft, also indicate the ability of these compounds to completely prevent or inhibit graft rejection. The results obtained in the standard pharmacological test with the use of adjuvant arthritis, also indicate the ability of compounds of the present invention to cure rheumatoid arthritis or delay its progression.

Based on the results of these standard pharmacological tests, we can conclude that these compounds are effective in the treatment or suppression of transplants, such as kidney, heart, liver, lung, bone marrow, pancreas (islet cells), cornea, small intestine, and skin allografts, and xenografts heart valves; in the treatment and submitting the NCI, rheumatoid arthritis, diabetes, myasthenia gravis and multiple sclerosis, and inflammatory diseases such as psoriasis, dermatitis, eczema, seborrhea, inflammatory bowel disease, lung inflammation (including asthma, chronic obstructive pulmonary disease, emphysema, acute respiratory distress syndrome, bronchitis and other similar diseases and uveitis.

Thanks to the detected mode of action, the compounds of the present invention can be considered to have antitumor, antifungal, and antiproliferative activity. It follows that the compounds of the present invention is also applicable in the treatment of solid tumors, T-cell leukemia/lymphoma in adults, mycosis and Hyper-proliferative vascular diseases such as restenosis and atherosclerosis. In the case of use in the treatment of restenosis, the compounds of the present invention is preferably used for the treatment of restenosis occurring after plastic surgery on the blood vessels. If used for this purpose, the compounds of the present invention can be administered prior to surgery, during surgery, after surgery or when any combination of the above conditions.

It is assumed that in those cases where the compounds of the present invention are used as immunosuppressive or anti-inflammatory drugs, they can be administered in combination with other one or more immunoregulatory agents. These immunoregulatory agents include (but not exhaustive list of possible): azathioprine, corticosteroids, such as prednisone and methylprednisolone, cyclophosphamide, rapamycin, cyclosporin a, FK-506, OCT-3, and ATG (antithymocyte globulin). If the compounds of the present invention are used in combination with such other drugs or agents for inducing immunosuppression or treating inflammatory conditions, to achieve the desired effect requires a smaller quantity of each of these tools. The rationale of this combinatorial therapy was given Stepkowski (Stepkowski), the results of studies which have shown that the use of a combination of rapamycin and cyclosporine a in subtherapeutic doses significantly extends the pot life of the allograft heart /Transplantation Proc. 23: 507 (1991)].

A solid carrier can include one or more substances which may also perform the function corrigentov, lubricating substances, substances that increase the solubility, suspendida agents, fillers, moving substances that improve the pressing means, a binder or dezintegriruetsja pill substances; it can also be an encapsulating material. In powders, the carrier is a finely powdered solid substance that is mixed with finely ground active ingredient. In tablets, the active ingredient is mixed in the correct proportions with the carrier having the necessary compression properties, and pressed into the desired shape of the required size. Preferably, the powders and tablets contain up to 99% of active ingredient. To the appropriate solid media include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, celluclay.

Liquid media used for preparation of solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives, for example, soljubilizatory, emulsifiers, buffers, preservatives, sweeteners and chemicals, altering the taste and smell of the drug, suspendresume substances, thickeners, dyes, viscosity regulators, stabilizers or osmoregulatory. Examples of acceptable liquid carriers for oral or parenteral administration include water (partially containing the above-mentioned additives such as cellulose derivatives, preferably, a solution of sodium carboxymethyl cellulose), alcohols (including monohydroxy alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, lecithins, and oils (e.g. fractionated coconut oil and peanut butter). For parenteral administration the carrier can also serve as oil ether, such as etiloleat and isopropylmyristate. Article is the introduction. As a liquid carrier for the compositions under pressure, can be used halogenated hydrocarbon or other pharmaceutically acceptable gas propellant is used.

Liquid pharmaceutical compositions in the form of sterile solutions or suspensions can be administered, for example, by intramuscular, intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously. Liquid or solid dosage form of the compounds may also be administered orally.

Compounds of the present invention can be administered rectally in the form of a conventional suppository. For administration by intranasal or intrabronchial inhalation or insufflation, the compounds of the present invention can be prepared in the form of aqueous or partially aqueous solution, which can then be used in the form of an aerosol. Compounds of the present invention can also be administered transdermally through the use of transdermal patch containing the active compound and a carrier, inert to the active compound, is non-toxic to the skin and providing systemic absorption of the drug into the bloodstream through the skin. The carrier may be in any of the many is to be viscous, liquid or semisolid emulsions of the type oil-in-water" type "water in oil". Can also be used pastes consisting of absorptive powders dispersed in petroleum jelly or hydrophilic petrolatum containing the active ingredient. There are a variety of options occlusal devices that can be used to extract the active ingredient into the bloodstream, for example, a semi-permeable membrane covering a reservoir containing the active ingredient with the carrier or without a carrier, or a matrix containing the active ingredient. In the literature there are other occlusal appliances.

In addition, the compounds of the present invention can be used in the form of a solution, cream, or lotion which has in its composition pharmaceutically acceptable carriers, containing 0.1-5%, preferably 2%, of active compounds that can be applied to the area affected by the fungus.

The dosage requirements vary according to the specific compositions, method of administration, the severity of present symptoms and specific object of treatment. According to the results obtained in the standard pharmacological tests, the estimated daily dose of the active is,01-5 mg/kg Typically, treatment begins with the introduction of low doses that are lower than the optimum dose of the compound. From this point, the dose is increased until the optimum effect, it is possible under these conditions, accurate dosages for oral, parenteral, nasal or intrabronchial injection determined by your doctor based on the experience of treatment of an individual patient. The pharmaceutical composition preferably is in the form of a unit dose, for example, in the form of tablets or capsules. In this form, the composition is divided into unit doses containing appropriate quantities of the active ingredient, forms a single dose can be composition in packages, for example, powders, sachets, ampoules, pre-filled syringes or sachets containing liquids. Medicinal form of a unit dose may be, for example, the capsule or tablet or it can be the appropriate number of any such compositions in Packed form.

The following examples illustrate the obtaining and biological activity of typical compounds of the present invention.

Example 1

31-O-(Triethylsilyl)-42-oxalipatin

Stage A. 31-O-(Triethylsilyl) rapamycin

To a solution of replylisteners.elementat (10 g, 37,88 mmol) within 30 min the resulting mixture was stirred at 0oC for 90 min and filtered. The filtrate was diluted with ethyl acetate (500 ml), washed with water (3x250 ml) and brine (CH ml), dried (MgSO4) and evaporated to dryness. The resulting material was re-dissolved in anhydrous THF (40 ml), cooled to 0oC and was treated with glacial acetic acid, cooled to the temperature of ice (150 ml) and water (80 ml). The mixture was stirred for 3 h at 0oC, was diluted with ethyl acetate (500 ml) and carefully brought to pH 7-8 NaHCO3at 0oC. the Organic layer was washed with water (2x250 ml), brine (CH ml), dried (MgSO4) and evaporated to dryness to obtain the product specified in the header with a quantitative yield

1H NMR (CDCl3, 400 MHz): of 1.66 (3H,6-CH3C=C), OF 1.75 (3H,30-CH3C=C), 3,14 (3H,41-OCH3), WITH 3.27 (3H,7-CH3O), 3,40 (3H,32-CH3O) of 4.12 (m, 1H,31-CH).

MS (neg. ion FAB, m/z): 1027,4 [M]-, 589,3.

Stage B. 31-O-(Triethylsilyl)-42-oxalipatin

A solution of 31-O-(triethylsilyl)rapamycin (17,32 g of 16.84 mmol) and periodinane dess-Martin (8.65 g, 20,35 mmol) in anhydrous dichloromethane (150 ml) was stirred under nitrogen atmosphere for 5 hours the mixture was filtered, the filtrate was diluted with ethyl acetate (50 the ial group pre-absorbed on a column of silica gel Merck-60 and instantly evaporated with hexane-ethyl acetate 95:5 and 7:2, obtaining a pure product specified in the header, with 64% yield.

1H NMR (CDCl3, 400 MHz): 0,42-of 0.48 (m, N), 0,80-of 0.83 (m, 6H), to 1.61 (3H, 6-CH3C= C), OR 1.77 (3H,30-CH3C=C), TOTALING 3.04 (3H,41-OCH3), AND 3.16 (3H,7-OCH3), OR 3.28 (3H,32-CH3O), 3,90 (m, 1H,41-CH).

MS (neg. ion FAB, m/z): 1025,3 [M]-.

Example 2

42 Deoxo-42-(hydroxyimino) rapamycin

The method of obtaining AND

A mixture of 42-oxalipatin (0,183 g, 0.22 mmol, obtained according to the method described in U.S. Patent 5023263), hydroxylamine hydrochloride (0,0143 g, 0.22 mmol) and sodium acetate (0.025 g, 0.3 mmol) in methanol (5 ml) was stirred under nitrogen atmosphere for 15 minutes the Mixture was evaporated to dryness and the residue was purified using flash chromatography (Merck silica gel 60, eluent 50% THF in hexane). Pure fractions were combined, evaporated and the resulting oil was recrystallized from isopropyl ether/cyclohexane at a ratio of 20: 80, with the receipt specified in the header of the product as a mixture of E/Z isomers (0.075 g, 40% yield).

1H NMR (CDCl3, 400 MHz): 1,65 (3H,6-CH3C=C), OF 1.75 (3H,30-CH3C=C), OF 3.13 (3H,41-OCH3), TO 3.34 (3H,7-OCH3), TO 3.41 (3H,32-OCH3).

13C NMR (CDCl3, 400 MHz): 215,28; 208,17; 169,26; 166,72; 158,83; 140,60; 140,07; 135,95; 135,67, 133,56; 130,18; 129,47; 126,62; 126,43;,92; 27,21; 27,05; 26,89; 25,27; 22,84; 21,97; 21,62; 21,46; 20,64; 16,32; 16,22; 16,09; 15,99; 14,74; 13,60; 13,18; 13,10; 10,33; 10,15.

MS (neg. ion FAB, m/z): 926 [M]-, 590, 334.

Analysis: Calculated for C51H78N2O13: 66,07; H 8,48; N TO 3.02.

Found: C 66,25; H 8,67; N 3,03.

The method of obtaining B

Stage A. 31-O-(Triethylsilyl)-42-deoxo-42-(hydroxyimino) rapamycin

Under anhydrous conditions a mixture of 31-O-(triethylsilyl)-42-oxalipatin example 1 (0,105 g is 0.102 mmol), hydroxylamine hydrochloride (7,6 mg, 0,109 mmol) and sodium acetate (12.5 mg, 0,153 mmol) in anhydrous methanol (5 ml) was stirred for 30 minutes the Mixture was filtered and the resulting filtrate was evaporated to dryness. The residue was re-dissolved in ethyl acetate (50 ml), washed with water (I ml) and brine (CH ml), dried (MgSO4) and evaporated to dryness to obtain specified in the header of the product as a mixture of E/Z isomers (0,114 g, 94% yield).

1H NMR (CDCl3, 400 MHz): 0,47-0,55 (m, N), 0,84-to 0.88 (m, 6H), of 1.65 (3H, 6-CH3C= C), OF 1.75 (3H,30-CH3C=C), 3,14 (3H, 41-OCH3), 3,26 (3H, 7-OCH3), 3,44 (3H, 32-CH3O).

MS (neg. ion FAB, M/z): 1040,7 [M]-.

Stage B. 42 Deoxo-42-(hydroxyimino)rapamycin

A solution of 31-O-(triethylsilyl)-42-deoxo-42-(hydroxyimino)rapamycin (1 g, 0,974 molpro 0oC. the Solution was diluted with ethyl acetate and extinguished 5% aqueous NaHCO3. The organic layer was washed with water, brine, dried (MgSO4) and evaporated to dryness, obtaining specified in the header of the product, identical to the material described in the method of obtaining A (0,812 g, yield of 89.9%).

The results obtained in the standard pharmacological tests:

LAF IC50: 3,88 nmol.

LAF: 0,85.

Time viability of skin graft: 10,80,4.

Example 3

42 deoxo-42-(hydroxyimino) rapamycin

To a solution of 31-O-(triethylsilyl)-42-deoxo-42-(hydroxyimino)rapamycin example 2, the Method of obtaining B, phase A (2,08 g, 2 mmol) in anhydrous methanol (75 ml) under nitrogen atmosphere at 0oC were simultaneously added within 30 min to 1 N. the solution cyanoborohydride sodium in tetrahydrofuran (2 ml) and 4 n HCl solution in dioxane so as to maintain the pH at 3.5. The mixture was stirred for 30 min, diluted with EtOAc and washed with 2.5% NaHCO3(100 ml), water (2x250 ml) and brine (CH ml), dried (MgSO4) and evaporated to dryness to obtain specified in the header of the product as a mixture of isomers (0,763 g, 41% yield).

1H NMR (CDCl3, 400 MHz): to 1.60 and 1.63 (3H, 6-CH3C=C), 1,72, and of 1.74 (3H, 30-, 590,3, 336,6.

Analysis. Calculated for C51H80N2O13: C 65,92, H 8,68, N 3,01.

Found: C 65,36; H 8,53; N 2,82.

The results obtained in the standard pharmacological test:

LAF IC50: the ceiling of 5.60 nmol

LAF: 0,18

Time viability of skin graft: 9,60,0

The percentage change in the group with adjuvant arthritis in comparison with the control group: -84%

Example 4

42-Deoxy-42-oxalipatin-42-0-carbamoyloximes

A mixture of 42 deoxo-42-(oxyimino)rapamycin example 2 (0,813 g, 0,876 mmol), sodium cyanate (0,228 g, 3,5), glacial acetic acid (8 ml) and water (8 ml) was stirred for 1.5 h under nitrogen atmosphere. The mixture was diluted with ethyl acetate (100 ml) and extinguished aqueous NaHCO3. The organic layer was washed with water, brine, dried (MgSO4) and evaporated to dryness. The crude product was dissolved in dichloromethane, pre-absorbed on silica gel Merck 60, and was purified using flash chromatography stepped gradient from 50% ethyl acetate in hexane to pure ethyl acetate to obtain specified in the header of the product as a mixture of E/Z isomers (0,289 g, 34% yield).

1NMR (CDCl3, 400 MHz): of 1.66 (3H,6-CH3C=C), OF 1.75 (3H, 30-CH3C=C), 3,14 (3H, 41-OCH3

MS (neg. ion FAB, m/z): 969,8 [M]-, 925,8 [M-CONH2]-590,6.

The results obtained in the standard pharmacological test:

LAF IC50: 2.00 nmol.

LAF: 0,25

Time viability of skin graft: 9,5 1,1

Example 5

a 42-[O-(Pyridine-2-ylmethyl)]-oxime 42-deoxy-42-oxalipatin

The connection specified in the header, obtained according to the method of obtaining B of example 2, replacing hydrochloride hydroxylamine hydrochloride O-(pyridine-2-ylmethyl)-hydroxylamine (output 71,1%).

1H NMR (CDCl3, 400 MHz): 1,654 (3H, 6-CH3C=C), 1,751 (3H, 30-CH3C=C) 3,138 (3H, 41-OCH3), 3,334 (3H, 7-OCH3), 3,338 (3H, 32-OCH3), 7,17-8,58 (mm, 4H, Harom).

13C NMR (CDCl3, 400 MHz): 1,58,96 (42-C=NO-).

LAF: 0,40

Time viability of skin graft: 7,8 0,8

The percentage change in the group with adjuvant arthritis in comparison with the control group: -53%.

Example 6

a 42-[O-(Pyridine-4-ylmethyl)]-oxime 42-deoxy-42-oxalipatin

The connection specified in the header, obtained according to the Method of obtaining B of example 2, replacing hydroxylamine hydrochloride the hydrochloride of O-(pyridine-4-ylmethyl)- hydroxylamine (output 34,5%).

1H NMR (CDCl3, 400 MHz): 1,653 (3H, 6-CH3C=C), 1,753 (3H, 30-CH3C=C) 3,153 (3H, 41-OCH3), 3,29 (HN, 7-OCH3and 32-OCH3), to 7.2 (m, 2H, Harom), charged 8.52 (m, 2H, Harom).

MS (neg. ion FAB, m/z): 1017,2 [M]-, 590,2, 425,1.

The results obtained in the standard pharmacological test:

LAP IC50: 1,80 nmol

LAF ratio: 0,31

Time viability of skin graft: 8,00,9

Example 7

a 42-[O-(tert-butyl)]-oxime 42-deoxy-42-oxalipatin

The connection specified in the header, obtained according to the Method of obtaining B of example 2, except for the replacement of the hydrochloride of hydroxylamine hydrochloride O-(tert-butyl)- hydroxylamine (output 26,6%).

1H NMR (CDCl3, 400 MHz): 1.28 (in N, Tr">

13C NMR (CDCl3, 400 MHz): 155,89 (42-C=NO-).

MS (neg. ion FAB, m/z): 982,5 [M]-, 590,3, 390,2.

The results obtained in the standard pharmacological test:

LAF: 49% inhibition at 0.1 Microm.

Example 8

a 42-[O-(phenylmethyl)]-oxime 42-deoxy-42 - oxalipatin

The connection specified in the header, obtained according to the Method of obtaining B of example 2, except for the replacement of the hydrochloride of hydroxylamine hydrochloride O-(phenylmethyl)-hydroxylamine (output 29,9%).

1H NMR (CDCl3, 400 MHz): 1,652 (3H, 6-CH3C=C), 1,747 (3H, 30-CH3C=C) 3,136 (3H, 41-OCH3), 3,332 (3H, 7-OCH3), TO 3.35 (3H, 32 - OCH3), 5,134 (2H, = NOCH2- at C-42), 7,27-7,37 (m, 5H, Harom).

13C NMR (CDCl3, 400 MHz): 158,48 (42-C=NO-).

MS (neg. ion FAB, m/z): 1016,2 [M]-, 590,1, 424,1.

The results obtained in the standard pharmacological tests:

LAF IC50: 21,67 nmol.

LAF: 0,03.

Example 9

a 42-[O-(allyl)]-oxime 42-deoxy-42-oxalipatin

The connection specified in the header, obtained according to the Method of obtaining B of example 2, except for the replacement of the hydrochloride of hydroxylamine hydrochloride O-(allyl)-hydroxylamine (output 57,1%).

1H NMR (CDCl2
C= at C-42), 5,17-5,31 (m, 2H, -C=CH2at C-42), 5,94-6,03 (m, 1H, -CCH=C-at C-42).

13C NMR (CDCl3, 400 MHz): 158,1 (42-C=N-0-).

MS (neg. ion FAB, m/z): 996,5 [M]-, 590,3, 374,2.

The results obtained in the standard pharmacological test:

LAF: 43% inhibition at 0.1 Microm.

Example 10

a 42-[O-(prop-2-inyl)]-oxime 42-deoxy-42-oxalipatin

The connection specified in the header, obtained according to the Method of obtaining B of example 2, except for the replacement of the hydrochloride of hydroxylamine hydrochloride O-(prop-2-inyl)- hydroxylamine (output 35,4%).

1H NMR (CDCl3, 400 MHz): of 1.66 (3H, 6-CH3C=C), OF 1.75 (3H, 30-CH3C=C), OF 3.13 (3H, 41-OCH3), TO 3.33 (3H, 7-OCH3), 3,415 (3H, 32-OCH3), 4,69 (2H, = NOCH2C, at C-42).

13C NMR (CDCl3, 400 MHz): 159,5 (42-C=NO-).

MC (neg. ion FAB, m/z): 964,2 [M]-, 590,2, 372,1.

The results obtained in the standard pharmacological test:

LAF IC50: 8,13 nmol.

LAF: 0,08.

Time viability of skin graft: 8,01,1

Example 11

42 Deoxo-42-[O-(pyridine-4-ylmethyl)]-hydroxylaminopurine

The connection specified in the header, obtained according to example 3, except sorapipatana example 6.

MC (neg. ion FAB, m/z): 1019,5 [M]-.

1. 42-Oximes and hydroxylamine rapamycin General formula I

< / BR>
in which X-Y is C=NOR1or CHNHOR2in which R1represents hydrogen, alkyl with 1 to 6 carbon atoms, alkenyl with 2 to 7 carbon atoms, quinil with 2 to 7 carbon atoms, a group of the formula -(CH2)mAr, in which Ar is phenyl or pyridinyl and m = 1 to 6; R2represents hydrogen or a radical of the formula -(CH2)mAr, in which Ar represents pyridinyl,

or farmatsevticheskii acceptable salt.

2. Connection on p. 1, in which X-Y is C=NOR1or its pharmaceutically acceptable salt.

3. Connection on p. 1, in which X-Y is a group CHNHOR2or its pharmaceutically acceptable salt.

4. Connection on p. 1 which is 42-deoxo-42-(hydroxyimino)rapamycin or its pharmaceutically acceptable salt.

5. Connection on p. 1 which is 42-deoxo-42-(hydroxyamino)rapamycin or its pharmaceutically acceptable salt.

6. Connection on p. 1 which is 42-deoxy-42-oxalipatin-42-O-carbamoyloximes or its pharmaceutically acceptable salt.

7. Connection on p. 1, which is Yu.

8. Connection on p. 1 which is 42-deoxy-42-oxalipatin-42-[O-(pyridine-4-ylmethyl)]-oxime or its pharmaceutically acceptable salt.

9. Connection on p. 1 which is 42-deoxy-42-oxalipatin-42-[O-(tert-butyl)]-oxime or its pharmaceutically acceptable salt.

10. Connection on p. 1 which is 42-deoxy-42-oxalipatin-42-[O-(phenylmethyl)]-oxime or its pharmaceutically acceptable salt.

11. Connection on p. 1 which is 42-deoxy-42-oxalipatin-42-(O-allyl)-oxime or its pharmaceutically acceptable salt.

12. Connection on p. 1 which is 42-deoxy-42-oxalipatin-42-[O-(prop-2-inyl)]-oxime or its pharmaceutically acceptable salt.

13. Connection on p. 1, which is 2-deoxo-42-[O-(pyridine-4-ylmethyl)]-hydroxylaminopurine or its pharmaceutically acceptable salt.

14. The method of treatment of a disease selected from the group consisting of transplant rejection or disease graft-versus-host and rheumatoid arthritis, in a mammal in need of treatment, which includes an introduction to the specified mammal is effective against diseases of the number of compounds having the structure

< / BR>
2)mAr, in which Ar is phenyl or pyridinyl and m = 1 to 6; R2represents hydrogen or a radical of the formula -(CH2)mAr, in which Ar represents pyridinyl,

or its pharmaceutically acceptable salt.

15. The pharmaceutical composition exhibiting immunosuppressive activity, which includes the connection patterns

< / BR>
in which X-Y is C= NOR1or CHNHOR2, R1represents hydrogen, alkyl with 1 to 6 carbon atoms, alkenyl with 2 to 7 carbon atoms, quinil with 2 to 7 carbon atoms, a group of the formula -(CH2)mAr, in which Ar is phenyl or pyridinyl and m = 1 to 6; R2represents hydrogen or a radical of the formula -(CH2)mAr, in which Ar represents pyridinyl,

or its pharmaceutically acceptable salt and a pharmaceutical carrier.

16. The connection patterns

< / BR>
in which R6is a group of SiR7R8R9in which each R7, R8and R9is independently alkyl with 1 to 8 carbon atoms.

17. Connection on p. 16, which is 31-O-(three-Atisreal)-42-oxalaplatin.

18. is the Qing (optionally protected hydroxyl functional group in position 31 silyl group of the formula R6defined in paragraph 16 with a hydroxylamine of the formula

H2NOR1,

in which R1has the above values,

with the formation of the corresponding oxime of the formula I, in which X-Y is a group With a=NOR1and destruction of specified protective group, R6.

 

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